Raman spectrometry appears to be an opportunity to perform rapid tests in microbiological diagnostics as it provides phenotype-related information from single bacterial cells thus holding the promise of direct analysis of clinical specimens without any time-consuming growth phase. Here, we demonstrate the feasibility of a rapid antibiotic-susceptibility determination based on the use of Raman spectra acquired on single bacterial cells. After a two-hour preculture step, one susceptible and two resistant E. coli strains were incubated, for only two hours, in the presence of different bactericidal antibiotics (gentamicin, ciprofloxacin, amoxicillin) in a range of concentrations that included the clinical breakpoints used as references in microbial diagnostic. Spectra were acquired and processed to isolate spectral modifications associated with the antibiotic effect. We evidenced an “antibiotic effect signature” which is expressed with specific Raman peaks and the coexistence of three spectral populations in the presence of antibiotic. We devised an algorithm and a test procedure that overcome single-cell heterogeneities to estimate the MIC and determinate the susceptibility phenotype of the tested bacteria using only a few single-cell spectra in four hours only if including the preculture step.

We present preliminary tests of hybrid pixel detectors consisting of the Medipix2 readout chip bump-bonded to a 1-mm-thick CdTe pixel detector. This room temperature imaging system for single photon counting has been developed within the Medipix2 European Collaboration for various imaging applications with X-rays and gamma rays, including dental radiography, mammography, synchrotron radiation, nuclear medicine, and radiation monitoring in nuclear facilities. The Medipix2 + CdTe hybrid detector features 256 256 square pixels, a pitch of 55 m, a sensitive area of 14 14 mm 2 . We analyzed the quality of the detector and bump-bonding and the response to nuclear radiation of the first CdTe hybrids. The CdTe pixel detectors, with Pt ohmic contacts, showed an ohmic response when negatively biased up to less than 60 V (electrons collection mode). Tests were also performed in holes collection mode, where a nonresistive behavior was observed above +15 V. We performed a series of imaging tests at low voltage bias with gamma radioactive sources and with an X-ray tube. Under uniform irradiation, we observed for all detectors the presence of numerous, stable structures in the form of small circles of about 200 m diameter, with the central pixels showing a reduced counting efficiency with respect to the periphery (in electrons counting regime). Also long filament structures have been observed. Further investigations will reveal whether they are due to an intrinsic detector response (e.g., due to Te inclusions) or to the bump-bonding process.

Abstract. Decreasing turnaround time is a paramount objective in clinical diagnosis. We evaluated the discrimination power of Raman spectroscopy when analyzing colonies from 80 strains belonging to nine bacterial and one yeast species directly on solid culture medium after 24-h (macrocolonies) and 6-h (microcolonies) incubation. This approach, that minimizes sample preparation and culture time, would allow resuming culture after identification to perform downstream antibiotic susceptibility testing. Correct identification rates measured for macrocolonies and microcolonies reached 94.1% and 91.5%, respectively, in a leave-one-strain-out cross-validation mode without any correction for possible medium interference. Large spectral differences were observed between macrocolonies and microcolonies, that were attributed to true biological differences. Our results, conducted on a very diversified panel of species and strains, were obtained by using simple and robust sample preparation and preprocessing procedures, while still confirming published results obtained by using more complex elaborated protocols. Instrumentation is simplified by the use of 532-nm laser excitation yielding a Raman signal in the visible range. It is, to our knowledge, the first side-by-side full classification study of microorganisms in the exponential and stationary phases confirming the excellent performance of Raman spectroscopy for early species-level identification of microorganisms directly from an agar culture. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.